The human ether-a-go-go-related gene (HERG) encodes an ion channel subunit underlying IKr, a potassium current required for the normal repolarization of ventricular cells in the human heart. More than 90 inherited mutations in HERG cause Long QT Syndrome (LQTS), a leading cause of sudden cardiac death. Some mutations alter gating, but more disrupt trafficking. Because the subunit composition of HERG is uncertain, and the mechanisms underlying HERG biogenesis, processing and targeting to the membrane are unknown, we carried out a yeast two-hybrid screen to identify proteins that interact with HERG. Using the carboxy terminus as bait to screen a human heart library, we isolated five genes encoding HERG-interacting proteins ("HIPs"). Two of these proteins have been previously identified: Tara, an actin-binding protein, and GM 130, a peripheral membrane protein of the Golgi apparatus. Little is known about the function of either. Tara co-localizes with HERG to a region in rat cardiac myocytes corresponding to the T-tubules, as determined by confocal immunocytochemistry. Consistent with a stabilizing role at the membrane, Tara enhances expression in HERG when co-expressed in Xenopus oocytes. GM 130 specifically localizes to the Golgi, where a prominent HERG signal is also observed. In contrast to Tara, GM130 suppresses HERG signal in oocytes. Deletion mapping in binary yeast two-hybrid assays reveals that the C terminus contains distinct domains with which the HIPs selectively interact. Certain LQT2 (HERG) mutations selectively disrupt interactions with only two of the proteins. Three of the proteins, Tara, H17 and H3, interact with each other, implying that they function as an interactive complex. Of the HIPs, Tara alone interacts with another cardiac ion channel protein, KvLQT1, in binary yeast two-hybrid assays, but none interacts with Shaker. Each HIP represents a potential target for LQTS to the extent that its expression is required for the normal expression or targeting of HERG channels. The long-range goal of this research is to elucidate the basic biological processes that are disrupted by the disease process. The specific aims of this proposal are: (1) to demonstrate that HERG and the HIPs interact in vivo: (2) to extend our immunocytochemical and electrophysiological analyses, tests for specificity and domain mapping; (3) to determine the necessity of HIP interactions for HERG channels by reciprocal analysis of HERG C terminal truncations and selective disruption of HIPs in native tissues and heterologous systems; and (4) to screen unmapped LQTS families for disease mutations in the genes encoding the HIPs.